The effect of insulin on S6 kinase activity in skeletal muscle from insulin sensitive and insulin resistant Pima Indians was examined. The S6 kinase was assayed in extracts of skeletal biopsies obtained from the vastus lateralis at 0, 15, 30, 45, 60, and 90 minutes during a hyperinsulinemic, (about 2000 muU/ml) euglycemic clamp. Prior to the infusion of insulin S6 kinase activity was similar in the groups. In insulin sensitive subjects, S6 kinase activity increased between 15 and 30 minutes of insulin infusion reaching a maximum at 45 minutes and then declined. S6 kinase activity also increased in resistant subjects, but the maximum occurred later, at 60 minutes, without the rapid increase between 15 and 30 minutes. The absence of an earlier insulin response of S6 kinase suggested that insulin resistant subjects may lack a particular earlier responding S6 kinase. Chromatography of extracts on FPLC MonoQ revealed two peaks of insulin-stimulated S6 kinase activity that eluded similarly in the two groups. Most of the increased activity occurred in peak 2 and immunoblot analysis revealed that the enzyme responsible for peak 1 activity is antigenically related to the 90 kilodalton S6 kinase and the peak 2 activity, is accounted for by an enzyme antigenically related to the 70 kilodalton S6 kinase. The results suggest that the mechanism for insulin resistance in Pima Indians occurs upstream from the S6 kinase in the pathway of insulin signal transduction. The project has been terminated.